Myelination is essential to the stability, structure and function of myelinated axons. Myelin enhances axon survival, regulates the axon cytoskeleton, impacts nodes of Ranvier, and inhibits axon regeneration. In each case, molecules on myelin engage complementary molecules on axons to initiate cell-to-cell signaling. Knowledge of the receptors and ligands responsible may provide insight into dysmyelinating disorders and help enhance axon regeneration. Myelin associated glycoprotein (MAG) participates in all of these effects of myelin on axons. Mice lacking MAG express myelin, but display axon degeneration, altered axon cytoskeleton, and altered nodes of Ranvier. MAG also inhibits axon regeneration. MAG binds to multiple ligands on the axon. Among these, gangliosides are implicated in several of MAG's effects. Mice lacking complex gangliosides display axon degeneration, altered axon cytoskeleton, and altered nodes of Ranvier. Gangliosides are also ligands for MAG-mediated inhibition of axon regeneration in vitro. Aim 1. The major MAG-binding gangliosides in nerve tissue, GDla and GT1b, are synthesized by addition of a sialic acid to the terminal galactose of non-MAG binding gangliosides GM1 and GD1b respectively. The sialyltransferases responsible have not been established, hi collaboration with Dr. Jamey Marth (UCSD), we propose a program to identify the genes responsible and construct a mouse strain lacking GDla and GTlb. This mouse will provide a definitive test of the role of these MAG ligands in vivo and cellular substrates for reconstitution of MAG function in vitro. Aim 2. Independent studies implicate gangliosides and the Nogo66 receptor, NgR, as independent MAG ligands. We will test whether different neurons use different functional MAG ligands, and whether they act independently or interactively, using cerebellar granule neurons, dorsal root ganglion neurons, and embryonic stem cell derived motoneurons in vitro. Aim 3. Clustering gangliosides is sufficient to initiate neuronal signaling, including RhoA activation and inhibition of axon outgrowth. Since gangliosides populate the outer leaflet of the plasma membrane, they must recruit other molecules to generate transmembrane signals. We will use cell surface ganglioside engineering in situ to identify ganglioside-associated proteins that may serve as proximal molecules in ganglioside-mediated signaling.